The DNA damage checkpoint delays progress of the cell cycle until damage to the genome is repaired. To understand the molecular basis of this surveillance mechanism in human cells, the P.I. laboratory has been studying Plk3, a member of the Polo family of protein kinases. Recent studies suggest that Plk3 is an important component of the DNA damage checkpoint machinery and that it targets both the mitotic activator Cdc25C and the tumor suppressor p53. The kinase activity of Plk3 is rapidly increased by exposure of cells to genotoxic stresses. Ectopic expression of an active form of Plk3 (Plk3-A) suppresses cell proliferation and induces apoptosis. Plk3 phosphorylates p53 in vitro on two major sites that includes serine-20, and a kinase-defective mutant of Plk3 (Plk3K52R) inhibits phosphorylation of p53 on serine-20 in vivo. Moreover, Plk3 concentrates at unduplicated centrosomes during the G1 phase of the cell cycle, and enforced expression of Plk3K52R results in centrosome amplification and the formation of multiple microtubule organization centers. Furthermore, human PLK3 localizes to chromosome 1p32, a locus thought to contain cancer susceptibility genes. The expression of PLK3 is down-regulated in human lung and head-neck carcinomas as well as in carcinogen-induced colon tumors in rats. On the basis of these various observations, we hypothesize that PLK3 is a tumor-suppressor gene whose product integrates signals that control genomic instability and achieves its effects, at least in part, through regulation of p53 and centrosome function. To test this hypothesis, the P.I.'s laboratory will (i) map all the phosphorylation sites of p53 targeted by Plk3 and determine the functional consequence of such phosphorylation; (ii) determine whether Plk3 acts immediately downstream of the protein kinases Chkl and Chk2 in the signaling pathways that underlie the cellular response to DNA damage; (iii) investigate whether Plk3 plays a role in the arrest of centrosome duplication in response to activation of the DNA damage checkpoint; and (iv) examine whether mice with a targeted disruption of PLK3 show an increase susceptibility to the development of colon, lung, or other cancers. The long-term goal of this project is to define mechanisms by which Polo family kinases function as key regulatory enzymes in the DNA damage checkpoint and by which dysregulation of these enzymes may result in genomic instability and neoplastic transformation.